CN114306591B - Liposome adjuvant, thermosensitive gel and thermosensitive gel preparation - Google Patents
Liposome adjuvant, thermosensitive gel and thermosensitive gel preparation Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 70
- 239000002671 adjuvant Substances 0.000 title claims abstract description 66
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 28
- 230000007704 transition Effects 0.000 claims abstract description 24
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- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 claims abstract description 15
- 239000002904 solvent Substances 0.000 claims abstract description 15
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims abstract description 14
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- 229960005486 vaccine Drugs 0.000 claims description 21
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- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
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Abstract
The application specifically discloses a liposome adjuvant, a temperature-sensitive gel and a temperature-sensitive gel preparation. The liposome adjuvant comprises 1-4 parts of DOTAP, 280-320 parts of soybean lecithin and 40-60 parts of cholesterol. The preparation method comprises the following steps: s1, dissolving DOTAP, soybean lecithin and cholesterol in a solvent; rotating and evaporating the solvent to obtain a film; s2, adding PBS into the film obtained in the step S1, stirring for 0.5-1.5h at 45-55 ℃, and dissolving the film to obtain liposome stock solution; and S3, extruding the liposome stock solution through a polycarbonate membrane for 20-80 times to obtain the liposome adjuvant. The temperature sensitive gel comprises poloxamer gel and liposome adjuvant; the temperature-sensitive gel can be used for preparing temperature-sensitive gel adjuvant. The liposome adjuvant has the advantages of adjusting the phase transition temperature and viscosity of the temperature-sensitive gel and improving the administration efficiency of the temperature-sensitive gel preparation.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a liposome adjuvant, a temperature-sensitive gel and a temperature-sensitive gel preparation.
Background
Currently approved vaccines (e.g., hand-foot-and-mouth vaccines) are mostly injections, which mainly induce strong systemic immune responses. However, most pathogens infect humans preferentially through mucosal surfaces of the gastrointestinal tract, respiratory tract, genitourinary tract, or eyes, and systemic vaccine delivery to infectious diseases, conventionally using injection, cannot induce a strong mucosal immune response.
Nasal cavities have a high density of dendritic cells that mediate strong systemic and mucosal immune responses against antigens and pathogens that invade the body through the upper respiratory tract and can avoid gastrointestinal damage when vaccines are administered through the nasal cavity; among them, nasal mucosa delivery is one of the technical routes to solve the shortcomings of existing products due to safety, efficacy, and ease of administration.
However, the absorption of drugs into nasal mucosa is limited, and how to improve the bioavailability of nasal mucosal administration is a problem that must be faced when studying nasal mucosal vaccines.
Disclosure of Invention
In order to increase the viscosity of the drug in nasal mucosa and improve the bioavailability of nasal mucosa administration, the application provides a liposome adjuvant, a temperature-sensitive gel and a temperature-sensitive gel preparation
In a first aspect, the present application provides a liposome adjuvant, which adopts the following technical scheme:
the liposome adjuvant comprises 1-4 parts of DOTAP, 280-320 parts of soybean lecithin and 40-60 parts of cholesterol.
Preferably, the liposome adjuvant comprises 3 parts of DOTAP, 297 parts of soybean lecithin and 50 parts of cholesterol in parts by weight.
The liposome adjuvant prepared by selecting soybean lecithin, DOTAP and cholesterol and proportioning the dosages of the soybean lecithin, DOTAP and cholesterol can be used for adjusting the phase transition temperature of temperature-sensitive gel and the viscosity after phase transition. So that the temperature-sensitive gel can act in the nasal cavity of the human body, and the temperature-sensitive gel solidifies after absorbing heat in the nasal cavity, thereby being attached to the nasal mucosa. When the temperature sensitive gel carries a medicament or vaccine, the medicament or vaccine can directly act on the nasal mucosa. The liposome adjuvant changes the phase transition temperature and viscosity of the temperature-sensitive gel, so that the effect of the medicament or vaccine on the nasal mucosa is improved.
In a second aspect, the present application provides a method of preparing a liposome adjuvant,
the preparation method of the liposome adjuvant comprises the following steps:
s1, dissolving DOTAP, soybean lecithin and cholesterol in a solvent; rotating and evaporating the solvent to obtain a film;
s2, adding PBS into the film obtained in the step S1, stirring for 0.5-1.5h at 45-55 ℃, and dissolving the film to obtain liposome stock solution;
and S3, extruding the liposome stock solution through a polycarbonate membrane for 20-80 times to obtain the liposome adjuvant. Preferably, the extrusion is performed 40 times.
The solvent may be at least one selected from methanol, ethanol, dichloromethane, and chloroform, and preferably, the solvent is ethanol.
The choice of different solvents has an effect on the film formation and the degree of hydration of the film in PBS:
when methylene chloride or dichloroethane is selected as a solvent, the obtained film is not uniform, precipitation occurs at the bottom of the bottle, and when methanol is selected as a solvent, the obtained film is wrinkled; when ethanol is selected as the solvent, the film area is large and the film is uniformly distributed at the bottom of the bottle.
For the hydration degree, when the solvent is ethanol, the liquid after hydration is milky white, which indicates that the film has good hydration effect and uniform distribution, and other solvents are selected, so that the liquid after hydration is more turbid.
In summary, when the solvent is ethanol, the film obtained after rotary evaporation and the hydration effect of the film are superior to those of other solvents.
Preferably, the polycarbonate membrane has a pore size of 200nm.
After extrusion through the polycarbonate membrane, the liposome adjuvant is homogenized to an average particle size of between 200 and 300nm, preferably 240 to 270nm.
The average particle size of the liposome is controlled to 240-270nm by selecting the specification of the polycarbonate membrane and controlling the extrusion times, so that the stability of the liposome adjuvant is improved, and the condition that the performance is influenced by coagulation of the liposome adjuvant after storage is reduced.
In a third aspect, the present application provides a temperature-sensitive gel, which adopts the following technical scheme,
the temperature-sensitive gel comprises 50-70 parts of poloxamer gel and 35-105 parts of liposome adjuvant in parts by weight;
the liposome adjuvant is the liposome adjuvant;
the phase transition temperature of the temperature-sensitive gel is 31-35 ℃.
When the temperature-sensitive gel is used as a temperature-sensitive gel preparation for nasal delivery, the phase transition temperature is preferably 31-35 ℃. The temperature of the nasal cavity of the human body is slightly lower than the body temperature under the influence of environmental factors, and when the phase transition temperature of the temperature-sensitive gel is selected to be 31-35 ℃, the temperature-sensitive gel can be quickly solidified in the nasal cavity. If the phase transition temperature of the temperature-sensitive gel is too low, the administration is not easy, and the temperature-sensitive gel is stored and transported. When the phase transition temperature of the temperature-sensitive gel is too high, the temperature-sensitive gel is not easy to change phase after being injected into the nasal cavity, so that the temperature-sensitive gel is difficult to adhere in the nasal cavity.
Preferably, the poloxamer gel consists of poloxamer 407 gel and poloxamer 118 gel, and the weight ratio of the poloxamer 407 gel to the poloxamer 118 gel is 1.5-2.5:1.
Preferably, the composition comprises 40 parts of poloxamer 407 gel, 20 parts of poloxamer 188 gel and 87.5 parts of liposome adjuvant.
In a fourth aspect, the present application claims a temperature-sensitive gel preparation, which adopts the following technical scheme.
A temperature-sensitive gel preparation comprises the temperature-sensitive gel.
Drugs or antigens can be added into the temperature-sensitive gel, so that a temperature-sensitive gel preparation can be prepared, and the temperature-sensitive gel preparation can be further used for drug delivery to nasal mucosa. Thereby improving the convenience of drug administration.
Preferably, the temperature-sensitive gel preparation further comprises an antigen and sucrose; the weight ratio of the sucrose to the temperature-sensitive gel is 10:80-180.
In a fifth aspect, the application claims the use of a temperature-sensitive gel formulation for the preparation of a vaccine for nasal mucosal delivery.
In summary, the present application has the following beneficial effects:
1. because the application selects soybean lecithin, DOTAP and cholesterol, and the dosage of the soybean lecithin, the DOTAP and the cholesterol is selected, the liposome adjuvant is obtained, and can be used for adjusting the phase transition temperature of gel, so that the phase transition temperature of temperature-sensitive gel is adapted to the temperature of human body, and the temperature-sensitive gel can be stably administered to nasal mucosa after the action of the temperature-sensitive gel and the nasal mucosa of the human body, thereby improving the drug effect.
2. According to the application, the phase transition temperature and the viscosity after phase transition of the thermosensitive gel are further optimized by adjusting the dosage relation of the liposome adjuvant in the thermosensitive gel, so that the adhesiveness of the thermosensitive gel is improved.
Detailed Description
Preparation of liposome adjuvant
The preparation examples 1 to 4 and the comparative preparation examples 1 to 3 were different in the amounts of the respective components, and the amounts of the respective components of the preparation examples 1 to 4 and the comparative preparation examples 1 to 3 are shown in Table 1.
Table 1 liposome adjuvant preparation examples 1-4 composition ratios
| Soybean lecithin/mg | DOTAP/mg | Cholesterol/mg | |
| Preparation example 1 | 299 | 1 | 50 |
| Preparation example 2 | 298 | 2 | 50 |
| Preparation example 3 | 297 | 3 | 50 |
| Preparation example 4 | 296 | 4 | 50 |
| Comparative preparation example 1 | 300 | 0 | 50 |
| Comparative preparation example 2 | 295 | 5 | 50 |
| Comparative preparation example 3 | 297 | 3 | 0 |
The liposome adjuvants of preparation examples 1-4 and comparative preparation examples 1-3 were prepared by the following methods
Preparation method of liposome adjuvant
S1, adding soybean lecithin, DPTAP and cholesterol into a eggplant-shaped bottle according to the formula amount, adding absolute ethyl alcohol for dissolution, and performing rotary evaporation at 60 ℃; and (3) evaporating absolute ethyl alcohol in the eggplant-shaped bottle to dryness, and forming a film in the eggplant-shaped bottle.
S2, adding 10ml of PBS into an eggplant-shaped bottle with a film on the wall of the bottle, and stirring for 1h at 50 ℃ to dissolve the film in the PBS to obtain liposome stock solution.
S3, transferring the liposome stock solution into a hermetic injector, and extruding the liposome stock solution through a polycarbonate membrane for 40 times to obtain the liposome adjuvant, wherein the aperture of the polycarbonate membrane is 200nm.
Comparative preparation example 4
The difference from preparation example 3 is that the pore diameter of the polycarbonate film in this comparative preparation example is 300nm.
The particle size of the liposome in the liposome adjuvant obtained in preparation examples 1 to 4 and comparative preparation examples 1 to 3 was examined for Zeta potential.
Measurement of liposome particle size: using a laser particle size analyzer (manufacturer model: bruceid 90Plus PALS), 1mL of liposome adjuvant was added to the assay vessel and each sample was automatically assayed three times.
Measurement of Zeta potential: and (3) measuring by adopting a dynamic light scattering particle size analyzer with a surface potential analysis function, adding 1mL of diluted nanoparticle suspension into a potential measuring sample cell, measuring three times in parallel, and taking an average value as a final result.
The measurement results of the particle size and Zeta potential are shown in Table 2
TABLE 2 particle size of liposomes and zeta potential detection
| Particle size (nm) | Zeta potential/mV | |
| Preparation example 1 | 241.2 | 35.23 |
| Preparation example 2 | 253.3 | 37.76 |
| Preparation example 3 | 264.3 | 42.34 |
| Preparation example 4 | 251.5 | 46.24 |
| Comparative preparation example 1 | 223.1 | 28.66 |
| Comparative preparation example 2 | 245.4 | 56.67 |
| Comparative preparation example 3 | 110.5 | 35.37 |
| Comparative preparation example 4 | 372.3 | 23.78 |
In Table 2, it can be seen that the average particle size of the liposome can be 240-270mm and the zeta potential can be 30-50mV in the liposome adjuvant prepared by selecting soybean lecithin, DOTAP and cholesterol for combination. The average particle size of the liposome is maintained at 200-300mm, especially 240-270mm, so that the stability of the liposome adjuvant can be improved, and when the particle size of the liposome adjuvant is too large, the liposome adjuvant is easy to be layered, and when the particle size of the liposome adjuvant is too small, the coating capacity of DOTAP can be influenced, so that the activity of the liposome adjuvant is reduced.
An increase in Zeta potential is advantageous for improving the stability of the liposome adjuvant, whereas if Zeta potential is too large, stimulation of cells is likely to occur.
Therefore, the liposome adjuvant obtained in preparation examples 1-4 has the advantages of high stability and small irritation to cells.
Preparation of temperature-sensitive gel
The temperature-sensitive gels of examples 1-3 and comparative examples 1, 2 were prepared as follows
Preparation method of temperature-sensitive gel
Dissolving 40mg of poloxamer 407 gel and 20mg of poloxamer 118 gel in 10ml of PBS to obtain a temperature-sensitive gel precursor solution, adding a liposome adjuvant into the temperature-sensitive gel precursor solution, and uniformly mixing to obtain temperature-sensitive gel.
The volume ratio of the temperature-sensitive gel precursor solution to the liposome adjuvant is 100:10.
Examples 1 to 3 and comparative examples 1 and 2 are different in that the liposome adjuvant used in the thermosensitive gel is different in the correspondence relationship between the thermosensitive gel and the liposome adjuvant as shown in Table 3
TABLE 3 correspondence between temperature-sensitive gel and liposome adjuvant in examples 1-3, comparative examples 1, 2
| Temperature sensitive gel | Liposome adjuvant |
| Example 1 | Preparation example 2 |
| Example 2 | Preparation example 3 |
| Example 3 | Preparation example 4 |
| Comparative example 1 | Comparative preparation example 1 |
| Comparative example 2 | Comparative preparation example 2 |
In examples 4 to 7 and comparative examples 3 and 4, the volume ratio of the thermosensitive gel precursor solution to the liposome adjuvant was different from that of example 2, and the other ratios and preparation methods were the same
TABLE 4 volume ratio of temperature sensitive gel precursor solutions to liposome adjuvants for examples 2, 4-7, comparative examples 3, 4
| Group of | Volume ratio of temperature-sensitive gel precursor solution to liposome adjuvant |
| Example 2 | 100:10 |
| Example 4 | 100:15 |
| Example 5 | 100:20 |
| Example 6 | 100:25 |
| Example 7 | 100:30 |
| Comparative example 3 | Without addition of liposome adjuvant |
| Comparative example 4 | 100:35 |
Temperature sensitive gel property detection
The temperature-sensitive gels prepared in examples 1 to 7 and comparative examples 1 to 4 were examined for their phase transition temperature (. Degree. C.) and viscosity (mPa.S)
The temperature-sensitive gel phase transition temperature is detected by adopting the following method:
and (3) refrigerating the temperature-sensitive gel at 4 ℃ for 2 hours, taking out the temperature-sensitive gel when detecting, adding a stirrer with the length of 0.5cm multiplied by 1.0cm, and regulating the stirring rotating speed to 150r/min. Heating in a water bath environment at a heating speed of 2 ℃/min. The temperature at which the stirrer stopped rotating was observed, the temperature at this time was defined as the gelation temperature, and the results were averaged by measuring 3 times in parallel for each group.
The viscosity of the temperature-sensitive gel was measured using a viscometer:
20ml of the prepared temperature-sensitive gel is placed in a beaker with the diameter not smaller than 70mm, and the temperature is kept in a constant-temperature water bath at 25 ℃ for 0.5h. The viscosity of the samples was measured using an NDJ-1 viscometer. The absolute viscosity of the solution is obtained by multiplying the reading indicated by the pointer on the dial plate by a specific coefficient on the coefficient table during measurement. Each group was assayed 3 times in parallel and the results averaged.
The test results are shown in Table 4.
Table 5 phase transition temperature, viscosity of temperature sensitive gel.
| Group of | Phase transition temperature (. Degree. C.) | Viscosity (mPa. S) |
| Example 1 | 32.5 | 210 |
| Example 2 | 32.1 | 235 |
| Example 3 | 33.4 | 202 |
| Example 4 | 33.7 | 254 |
| Example 5 | 34.1 | 278 |
| Example 6 | 34.3 | 293 |
| Example 7 | 34.5 | 252 |
| Comparative example 1 | 29.9 | 192 |
| Comparative example 2 | 30.6 | 187 |
| Comparative example 3 | 28.6 | 180 |
| Comparative example 4 | 35.3 | 202 |
In combination with table 5, for viscosity, the viscosity of the thermosensitive gel after phase transition should be 200-300mpa·s, when the viscosity of the thermosensitive gel is too low, the adhesion capability of the thermosensitive gel in the nasal cavity is easily reduced, the administration of the thermosensitive gel preparation to the nasal cavity is affected, and if the viscosity is too high, the nasal cavity is easily stimulated, and nasal secretion is promoted to increase, so that the administration effect is affected.
By combining the embodiment 2 and the comparative example 3, the phase transition temperature of the temperature-sensitive gel and the viscosity of the temperature-sensitive gel after phase transition can be obviously improved by adding the liposome adjuvant.
It can be seen from the combination of examples 1, 2 and 3, comparative examples 1 and 2 that the liposome adjuvant can significantly change the phase transition temperature of the thermosensitive gel and the obtained thermosensitive gel has higher viscosity when the weight ratio of DOTAP to lecithin is 3:297.
It can be seen from the combination of examples 2, 4, 5, 6, 7 and comparative example 4 that the phase transition temperature and viscosity of the temperature-sensitive gel can be adjusted by changing the amount of the liposome adjuvant, and the phase transition temperature of the temperature-sensitive gel is gradually increased and the viscosity is sequentially decreased as the amount of the liposome adjuvant is increased. The viscosity of the temperature sensitive gel was highest when the liposome adjuvant addition was 25%.
Preparation of temperature-sensitive gel preparation
Adding the antigen and the sucrose into the temperature-sensitive gel to obtain the temperature-sensitive gel preparation.
Specifically, the adding time of the antigen and the sucrose is before the mixing of the temperature-sensitive gel precursor solution and the liposome adjuvant: adding antigen and sucrose into the temperature-sensitive gel precursor solution according to the formula amount, uniformly mixing, and adding a liposome adjuvant to obtain the temperature-sensitive gel preparation carrying the temperature-sensitive gel antigen.
When the antigen is selected as the hand-foot-and-mouth antigen, the hand-foot-and-mouth vaccine temperature-sensitive gel preparation adopts the following preparation method:
dissolving 40mg of poloxamer 407 gel and 20mg of poloxamer 188 gel in 10ml of PBS to obtain a temperature-sensitive gel precursor solution, adding the antigen into the temperature-sensitive gel precursor solution according to the requirement that a target product contains 50U of hand-foot-mouth vaccine antigen, then adding 10mg of sucrose, and uniformly mixing; adding liposome adjuvant, and mixing to obtain hand-foot-mouth vaccine temperature-sensitive gel preparation.
The difference between application examples 1 to 7 is that the temperature-sensitive gels of examples 1 to 7 were selected, respectively;
comparative application examples 1 to 4 differ in that the temperature-sensitive gels of comparative examples 1 to 4 were selected, respectively;
the correspondence of the hand-foot-mouth vaccine temperature-sensitive gel preparation and the temperature-sensitive gel is shown in table 5.
TABLE 6 correspondence between hand-foot-mouth vaccine thermosensitive gel formulations and thermosensitive gels
| Hand-foot-mouth vaccine temperature-sensitive gel preparation | Temperature sensitive gel |
| Application example 1 | Example 1 |
| Application example 2 | Example 2 |
| Application example 3 | Example 3 |
| Application example 4 | Example 4 |
| Application example 5 | Example 5 |
| Application example 6 | Example 6 |
| Application example 7 | Example 7 |
| Comparative application example 1 | Comparative example 1 |
| Comparative application example 2 | Comparative example 2 |
| Comparative application example 3 | Comparative example 3 |
| Comparative application example 4 | Comparative example 4 |
Detection of hand-foot-and-mouth vaccine temperature-sensitive gel preparation effect mouse immunity: 10ul of the hand-foot-mouth vaccine temperature sensitive gel preparation is immunized 2 times through nasal mucosa, the interval is 2 weeks, and the orbital blood is taken 14 days after immunization to prepare serum; washing with 200ul physiological saline, collecting nasal mucus, removing impurities with 0.2um needle filter, and storing at-20deg.C; serum antibodies and intra-mucus antibody levels were determined by classical cytopathic methods.
The results are shown in Table 6
TABLE 7 serum antibody IgG concentration (μg/ml) and mucus IgA concentration (μg/ml).
| Group of | Serum IgG | Mucus IgA |
| Application example 1 | 160 | 32 |
| Application example 2 | 192 | 32 |
| Application example 3 | 160 | 32 |
| Application example 4 | 192 | 64 |
| Application example 5 | 192 | 64 |
| Application example 6 | 276 | 148 |
| Application example 7 | 256 | 128 |
| Comparative application example 1 | 128 | 16 |
| Comparative application example 2 | 64 | 8 |
| Comparative application example 3 | 128 | ＜8 |
| Comparative application example 4 | 65 | ＜8 |
It can be seen from the combination of Table 7 that the nasal delivery hand-foot-and-mouth vaccine temperature-sensitive gel preparation prepared by application examples 1-7 can significantly improve the serum antibody IgG and mucous antibody IgA levels in mice after acting on the mice. The serum antibody IgG concentration in application examples 1-7 can be above 160. Mu.g/ml, and most preferably 276. Mu.g/ml. There was a significant improvement over the comparative examples.
The change is probably due to the fact that the temperature-sensitive gel in the application example 6 can be attached to the nasal mucosa more stably, so that the effect of the hand-foot-mouth vaccine is better and more stable, and the liposome adjuvant has a slow release wrapping lease on the hand-foot-mouth antigen, so that the hand-foot-mouth antigen can act on the nasal mucosa more effectively, and the immune effect of the nasal delivery hand-foot-mouth vaccine temperature-sensitive gel preparation on mice is improved.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (4)
1. The temperature-sensitive gel is characterized by comprising 50-70 parts by weight of poloxamer gel and 35-105 parts by weight of liposome adjuvant;
the liposome adjuvant comprises 1-4 parts of DOTAP, 280-320 parts of soybean lecithin and 40-60 parts of cholesterol;
the poloxamer gel consists of poloxamer 407 gel and poloxamer 188 gel, and the weight ratio of the poloxamer 407 gel to the poloxamer 188 gel is 1.5-2.5:1;
the phase transition temperature of the temperature-sensitive gel is 31-35 ℃;
the preparation method of the liposome adjuvant comprises the following steps:
s1, dissolving DOTAP, soybean lecithin and cholesterol in ethanol; rotating and evaporating the solvent to obtain a film;
s2, adding PBS into the film obtained in the step S1, stirring for 0.5-1.5h at 45-55 ℃, and dissolving the film to obtain liposome stock solution;
and S3, passing the liposome stock solution through a polycarbonate membrane with the pore diameter of 200nm, and extruding for 20-80 times to obtain the liposome adjuvant.
2. A temperature-sensitive gel according to claim 1, wherein: the temperature-sensitive gel comprises 40 parts of poloxamer 407 gel, 20 parts of poloxamer 188 gel and 87.5 parts of liposome adjuvant according to parts by weight.
3. A temperature-sensitive gel preparation, which is characterized by comprising the temperature-sensitive gel according to any one of claims 1-2, a hand-foot-and-mouth antigen and sucrose; the weight ratio of the sucrose to the temperature-sensitive gel is 10:80-180.
4. Use of a temperature-sensitive gel formulation as claimed in claim 3 in the preparation of a hand-foot-and-mouth vaccine for delivery via nasal mucosa.
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